Protease inhibitors

ABSTRACT

This invention relates to the compound of Formula (I) or a pharmaceutically acceptable salt, hydrate or solvent thereof, which is an inhibitor of cysteine proteases, particularly cathespin K, and is useful in the treatment of diseases in which inhibition of bone loss or of cartilage degradation is a factor

This application is a 371 of PCT/US00/30757, field Nov. 8, 2000, whichclaims the benefit of Provisional Application 60/164,560, filed Nov. 10,1999.

FIELD OF THE INVENTION

This invention relates to a novel deuterated 4-amino-azepan-3-oneprotease inhibitor. This compound is particularly an inhibitor ofcysteine and serine proteases, more particularly an inhibitor ofcysteine proteases. The compound of this invention even moreparticularly inhibits cysteine proteases of the papain superfamily, andyet more particularly cysteine proteases of the cathepsin family. In themost preferred embodiment, this invention relates to a compound whichinhibits cathepsin K. Such compound is particularly useful for treatingdiseases in which cysteine proteases are implicated, especially diseasesof excessive bone or cartilage loss, e.g., osteoporosis, periodontitis,and arthritis.

BACKGROUND OF THE INVENTION

Cathepsin K is a member of the family of enzymes which are part of thepapain superfamily of cysteine proteases. Cathepsins B, H, L, N and Shave been described in the literature. Recently, cathepsin K polypeptideand the cDNA encoding such polypeptide were disclosed in U.S. Pat. No.5,501,969 (called cathepsin O therein). Cathepsin K has been recentlyexpressed, purified, and characterized. Bossard, M. J., et al., (1996)J. Biol. Chem. 271, 12517-12524; Drake, F. H., et al., (1996) J. Biol.Chem. 271, 12511-12516; Bromme, D., et al., (1996) J. Biol. Chem. 271,2126-2132.

Cathepsin K has been variously denoted as cathepsin O, cathepsin X orcathepsin O2 in the literature. The designation cathepsin K isconsidered to be the more appropriate one (name assigned by NomenclatureCommittee of the International Union of Biochemistry and MolecularBiology).

Cathepsins of the papain superfamily of cysteine proteases function inthe normal physiological process of protein degradation in animals,including humans, e.g., in the degradation of connective tissue.However, elevated levels of these enzymes in the body can result inpathological conditions leading to disease. Thus, cathepsins have beenimplicated in various disease states, including but not limited to,infections by pneumocystis carinii, trypsanoma cruzi, trypsanoma bruceibrucei, and Crithidia fusiculata; as well as in schistosomiasis malaria,tumor metastasis, metachromatic leukodystrophy, muscular dystrophy,amytrophy, and the like. See International Publication Number WO94/04172, published on Mar. 3, 1994, and references cited therein. Seealso European Patent Application EP 0 603 873 A1, and references citedtherein. Two bacterial cysteine proteases from P. gingivallis, calledgingipains, have been implicated in the pathogenesis of gingivitis.Potempa, J., et al. (1994) Perspectives in Drug Discovery and Design, 2,445-458.

Cathepsin K is believed to play a causative role in diseases ofexcessive bone or cartilage loss. Bone is composed of a protein matrixin which spindle- or plate-shaped crystals of hydroxyapatite areincorporated. Type I Collagen represents the major structural protein ofbone comprising approximately 90% of the structural protein. Theremaining 10% of matrix is composed of a number of non-collagenousproteins, including osteocalcin, proteoglycans, osteopontin,osteonectin, thrombospondin, fibronectin, and bone sialoprotein.Skeletal bone undergoes remodeling at discrete foci throughout life.These foci, or remodeling units, undergo a cycle consisting of a boneresorption phase followed by a phase of bone replacement.

Bone resorption is carried out by osteoclasts, which are multinuclearcells of hematopoietic lineage. The osteoclasts adhere to the bonesurface and form a tight sealing zone, followed by extensive membraneruffling on their apical (i.e., resorbing) surface. This creates anenclosed extracellular compartment on the bone surface that is acidifiedby proton pumps in the ruffled membrane, and into which the osteoclastsecretes proteolytic enzymes. The low pH of the compartment dissolveshydroxyapatite crystals at the bone surface, while the proteolyticenzymes digest the protein matrix. In this way, a resorption lacuna, orpit, is formed. At the end of this phase of the cycle, osteoblasts laydown a new protein matrix that is subsequently mineralized. In severaldisease states, such as osteoporosis and Paget's disease, the normalbalance between bone resorption and formation is disrupted, and there isa net loss of bone at each cycle. Ultimately, this leads to weakening ofthe bone and may result in increased fracture risk with minimal trauma.

The abundant selective expression of cathepsin K in osteoclasts stronglysuggests that this enzyme is essential for bone resorption. Thus,selective inhibition of cathepsin K may provide an effective treatmentfor diseases of excessive bone loss, including, but not limited to,osteoporosis, gingival diseases such as gingivitis and periodontitis,Paget's disease, hypercalcemia of malignancy, and metabolic bonedisease. Cathepsin K levels have also been demonstrated to be elevatedin chondroclasts of osteoarthritic synovium. Thus, selective inhibitionof cathepsin K may also be useful for treating diseases of excessivecartilage or matrix degradation, including, but not limited to,osteoarthritis and rheumatoid arthritis. Metastatic neoplastic cellsalso typically express high levels of proteolytic enzymes that degradethe surrounding matrix. Thus, selective inhibition of cathepsin K mayalso be useful for treating certain neoplastic diseases.

It now has been discovered that a certain novel deuterated compound is aprotease inhibitor, most particularly an inhibitor of cathepsin K, andthat this compound is useful for treating diseases characterized by boneloss, such as osteoporosis and gingival diseases, such as gingivitis andperiodontitis, or by excessive cartilage or matrix degradation, such asosteoarthritis and rheumatoid arthritis.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a novel deuterated4-amino-azepan-3-one protease inhibitor, particularly an inhibitor ofcysteine and serine proteases. More particularly, the present inventionrelates to such a compound which inhibits cysteine proteases, and yetmore particularly cysteine proteases of the papain superfamily.Preferably, this invention relates to such a compound which inhibitscysteine proteases of the cathepsin family and most preferably, acompound which inhibits cathepsin K. The compound of the presentinvention is useful for treating diseases which may be therapeuticallymodified by altering the activity of such proteases.

Accordingly, in the first aspect, this invention provides a compound,quinoxaline-2-carboxylic acid{(S)-3-methyl-1-[(2,2′,4-trideuterio)-3-oxo-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide,according to Formula I:

In another aspect, this invention provides a pharmaceutical compositioncomprising a compound according to Formula I and a pharmaceuticallyacceptable carrier.

In yet another aspect, this invention provides a method of treatingdiseases in which the disease pathology may be therapeutically modifiedby inhibiting proteases, such as cysteine and serine proteases. Inparticular, the method includes treating diseases by inhibiting cysteineproteases, and particularly cysteine proteases of the papainsuperfamily. More particularly, the inhibition of cysteine proteases ofthe cathepsin family, such as cathepsin K is described.

In another aspect, the compound of this invention is especially usefulfor treating diseases characterized by bone loss, such as osteoporosis,and gingival diseases, such as gingivitis and periodontitis, or byexcessive cartilage or matrix degradation, such as osteoarthritis andrheumatoid arthritis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound, quinoxaline-2-carboxylic acid{(S)-3-methyl-1-[(2,2′,4-trideuterio)-3-oxo-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide,of Formula (I):

or a pharmaceutically acceptable salt, hydrate or solvate thereof.

The present invention includes all hydrates, solvates, complexes,polymorphs and prodrugs of the compound of Formula (I). Prodrugs are anycovalently bonded compounds which release the active parent drugaccording to Formula (I) in vivo. Prodrugs of the compound of thepresent invention include ketone derivatives, specifically ketals orhemiketals.

All forms of isomers resulting from the presence of a chiral center inthe inventive compound, including enantiomers and diastereomers, areintended to be covered herein. The inventive compound may be used as aracemic mixture, an enantiomerically enriched mixture, or the racemicmixture may be separated using well-known techniques and an individualenantiomer may be used alone.

In the event that the present compound may exist in tautomieric forms,such as keto-enol tautomers, each tautomeric form is contemplated asbeing included within this invention whether existing in equilibrium orpredominantly in one form.

Compared to the corresponding 5 and 6 membered ring compounds, the 7membered ring compound of the present invention is configurationallymore stable at the carbon center alpha to the ketone.

Definitions

Abbreviations and symbols commonly used in the peptide and chemical artsare used herein to describe the compounds of the present invention. Ingeneral, the amino acid abbreviations follow the IUPAC-IUB JointCommission on Biochemical Nomenclature as described in Eur. J. Biochem.,158, 9 (1984). In particular, throughout this application, m-CPBA meansmeta-chloroperoxybenzoic acid; Boc means tert-butoxycarbonyl; EDC means1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DMSO meansmethyl sulfoxide; and TEA means triethylamine.

Method of Preparation

The compound of the Formula (I) is generally prepared according toScheme 1. The individual diastereomers of quinoxaline-2-carboxylic acid{(S)-3-methyl-1-[(2,2′,4-trideuterio)3-oxo-1-(1-oxy-pyridine-2-sulfonyl)-azepan4-ylcarbamoyl]-butyl}amide10 and 11 may be prepared as outlined in Scheme 1. Alkylation ofallyl-carbamic acid benzyl ester (1) with 5-bromo-1-pentene in thepresence of a base such as sodium hydride provides the diene 2.Treatment of diene 2 with bis(tricyclohexylphosphine)benzylidineruthenium (IV) dichloride develpoed by Grubbs provides the2,3,4,7-tetrahydro-azepine-1-carboxylic acid benzyl ester 3. Epoxidationof azepine 3 may be effected with standard oxidizing agents common tothe art such as m-CPBA to provide epoxide 4. Nucleophilic epoxide ringopening of 4 may be effected with a reagent such as sodium azide toprovide

Reagents and Conditions: a.) NaH, 5-bromo-1-pentene, DMF; b.)bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride,CH₂Cl₂; c.) m-CPBA, CH₂Cl₂; d.) NaN₃, CH₃OH, H₂O, NH₄Cl; e.)1,3-propanedithiol, TEA, methanol; f.) N-Boc-leucine, EDC, CH₂Cl₂; g.)10% Pd/C, H₂; h.) 2-pyridinesulphonyl chloride-N-oxide, sat. NaHCO₃,CH₂Cl₂; i.) 4 N HCl/dioxane, methanol; j.) quinoxaline-2-carboxylicacid, EDC, CH₂Cl₂; k.) pyridine sulfur trioxide complex, DMSO, TEA; l.)CD₃OD;D₂O (10:1), TEA; m.) HPLC separation. the azido alcohol (notshown). The intermediate azido alcohol may be reduced to the aminoalcohol 5 under conditions common to the art such as 1,3-propanedithioland triethylamine in methanol or with triphenylphosphine intetrahydrofuran and water. Acylation of 5 may be effected with an acidsuch as N-Boc-leucine in the presence of a coupling agent such as EDC.Removal of the benzyloxycarbonyl protecting group with hydrogen gas inthe presence of 10% Pd/C provides the amine 6. Treatment of the amine 6with 2-pyridinesulphonylchloride-N-oxide in the presence of saturatedsodium bicarbonate and dichloromethane followed by removal of thetert-butoxycarbonyl protecting group under acidic conditions provides 7.Coupling of 7 with quinoxaline-2-carboxylic acid may be effected with acoupling agent such as EDC to provide intermediate alcohol 8. Alcohol 8may be oxidized with an oxidant such as sulfur trioxide pyridine complexin DMSO and triethylamine to provide the ketone 9 as a mixture ofdiastereomers. Treatment of ketone 9 with triethylamine in CD₃OD:D₂O atreflux provides the deuterated analog as a mixture of diastereomerswhich are separated by HPLC to provide the deuterated compounds 10 and11.

The starting materials used herein are commercially available or areprepared by routine methods well known to those of ordinary skill in theart and can be found in standard reference books, such as the COMPENDIUMOF ORGANIC SYNTHETIC METHODS, Vol. I-VI (published byWiley-Interscience).

Coupling methods to form amide bonds herein are generally well-known inthe art. The methods of peptide synthesis generally set forth byBodansky et al., THE PRACTICE OF PEPTIDE SYNTHESIS, Springer-Verlag,Berlin, 1984; E. Gross and J. Meienhofer, THE PEPTIDES, Vol. 1, 1-284(1979); and J. M. Stewart and J. D. Young, SOLID PHASE PEPTIDESYNTHESIS, 2d Ed., Pierce Chemical Co., Rockford, Ill., 1984, aregenerally illustrative of the technique and are incorporated herein byreference.

Synthetic methods useful in preparing the compound of this inventionfrequently employ protective groups to mask a reactive functionality orminimize unwanted side reactions. Such protective groups are describedgenerally in Green, T.W, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, JohnWiley & Sons, New York (1981). The term “amino protecting groups”generally refers to the Boc, acetyl, benzoyl, Fmoc and Cbz groups andderivatives thereof as known to the art. Methods for protection anddeprotection, and replacement of an amino protecting group with anothermoiety are well known.

Acid addition salts of the compound of Formula (I) are prepared in astandard manner in a suitable solvent from the parent compound and anexcess of an acid, such as hydrochloric, hydrobromic, hydrofluoric,sulfuric, phosphoric, acetic, trifluoroacetic, maleic, succinic ormethanesulfonic acid.

Novel Intermediate

The present invention also provides a novel intermediate,quinoxaline-2-carboxylic acid{(S)-3-methyl-1-[3-hydroxy-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide(8-Scheme-1), of Formula (II), useful in the synthesis of the compoundof Formula (I) according to Scheme 1.

Process for Synthesis of Inventive Compounds

Referring to Schemes 1 herein above, the present invention provides aprocess for the synthesis of compounds of Formula (1) comprising thestep of oxidizing the appropriate compound of Formula (II) with anoxidant to provide the compound of Formula (I) as a mixture ofdiastereomers. Preferably the oxidant is sulfur trioxide pyridinecomplex in DMSO and triethylamine.

Referring to Scheme 1, the present invention also provides a process forthe synthesis of deuterated compounds of Formula (I). Specifically, whena deuterated isomer is desired, an additional step, following theoxidation step, of deuterating the protonated isomer with a deuteratingagent to provide the deuterated compound of Formula (I) as a mixture ofdiastereomers is added to the synthesis. Preferably, the deuteratingagent is CD₃OD:D₂O (10:1) in triethylamine.

The process further comprises the step of separating the diasteromers ofFormula (I) by separating means, preferably by high presssure liquidchromatography (HPLC).

Utility of the Present Invention

The present compound exhibits superior chiral stability when compared tothe protonated isomer.

This invention also provides a pharmaceutical composition whichcomprises a compound according to Formula (I) and a pharmaceuticallyacceptable carrier, excipient or diluent. Accordingly, the compound ofFormula (I) may be used in the manufacture of a medicament.Pharmaceutical compositions of the compound of Formula (I) prepared ashereinbefore described may be formulated as solutions or lyophilizedpowders for parenteral administration. Powders may be reconstituted byaddition of a suitable diluent or other pharmaceutically acceptablecarrier prior to use. The liquid formulation may be a buffered,isotonic, aqueous solution. Examples of suitable diluents are normalisotonic saline solution, standard 5% dextrose in water, or bufferedsodium or ammonium acetate solution. Such formulation is especiallysuitable for parenteral administration, but may also be used for oraladministration or contained in a metered dose inhaler or nebulizer forinsulation. It may be desirable to add excipients such aspolyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethyleneglycol, mannitol, sodium chloride, or sodium citrate.

Alternately, this compound may be encapsulated, tableted, or prepared inan emulsion or syrup for oral administration. Pharmaceuticallyacceptable solid or liquid carriers may be added to enhance or stabilizethe composition, or to facilitate preparation of the composition. Solidcarriers include starch, lactose, calcium sulfate dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar orgelatin. Liquid carriers include syrup, peanut oil, olive oil, salineand water. The carrier may also include a sustained release materialsuch as glyceryl monostearate or glyceryl distearate, alone or with awax. The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulating, and compressing, when necessary,for tablet forms; or milling, mixing and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly orfilled into a soft gelatin capsule.

For rectal administration, the compound of this invention may also becombined with excipients such as cocoa butter, glycerin, gelatin orpolyethylene glycols and molded into a suppository.

The compound of Formula (I) is useful as a protease inhibitor,particularly as an inhibitor of cysteine and serine proteases, moreparticularly as an inhibitor of cysteine proteases, even moreparticularly as an inhibitor of cysteine proteases of the papainsuperfamily, yet more particularly as an inhibitor of cysteine proteasesof the cathepsin family, most particularly as an inhibitor of cathepsinK. The present invention also provides useful compositions andformulations of said compound, including pharmaceutical compositions andformulations of said compound.

The present compound is useful for treating diseases in which cysteineproteases are implicated, including infections by pneumocystis carinii,trypsanoma cruzi, trypsanoma brucei, and Crithidia fusiculata; as wellas in schistosomiasis, malaria, tumor metastasis, metachromaticleukodystrophy, muscular dystrophy, amytrophy; and especially diseasesin which cathepsin K is implicated, most particularly diseases ofexcessive bone or cartilage loss, including osteoporosis, gingivaldisease including gingivitis and periodontitis, arthritis, morespecifically, osteoarthritis and rheumatoid arthritis, Paget's disease;hypercalcemia of malignancy, and metabolic bone disease.

Metastatic neoplastic cells also typically express high levels ofproteolytic enzymes that degrade the surrounding matrix, and certaintumors and metastatic neoplasias may be effectively treated with thecompound of this invention.

The present invention also provides methods of treatment of diseasescaused by pathological levels of proteases, particularly cysteine andserine proteases, more particularly cysteine proteases, even moreparticularly cysteine proteases of the papain superfamily, yet moreparticularly cysteine proteases of the cathepsin family, which methodscomprise administering to an animal, particularly a mammal, mostparticularly a human in need thereof the compound of the presentinvention. The present invention especially provides methods oftreatment of diseases caused by pathological levels of cathepsin K,which methods comprise administering to an animal, particularly amammal, most particularly a human in need thereof, an inhibitor ofcathepsin K, including the compound of the present invention. Thepresent invention particularly provides methods for treating diseases inwhich cysteine proteases are implicated, including infections bypneumocystis carinii, trypsanoma cruzi, trypsanoma brucei, and Crithidiafusiculata; as well as in schistosomiasis, malaria, tumor metastasis,metachromatic leukodystrophy, muscular dystrophy, amytrophy, andespecially diseases in which cathepsin K is implicated, mostparticularly diseases of excessive bone or cartilage loss, includingosteoporosis, gingival disease including gingivitis and periodontitis,arthritis, more specifically, osteoarthritis and rheumatoid arthritis,Paget's disease, hypercalcemia of malignancy, and metabolic bonedisease.

This invention further provides a method for treating osteoporosis orinhibiting bone loss which comprises internal administration to apatient of an effective amount of the compound of Formula (I), alone orin combination with other inhibitors of bone resorption, such asbisphosphonates (i.e., allendronate), hormone replacement therapy,anti-estrogens, or calcitonin. In addition, treatment with the compoundof this invention and an anabolic agent, such as bone morphogenicprotein, iproflavone, may be used to prevent bone loss or to increasebone mass.

In accordance with this invention, an effective amount of the compoundof Formula (I) is administered to inhibit the protease implicated in aparticular condition or disease. Of course, this dosage amount willfurther be modified according to the type of administration of thecompound. For example, for acute therapy, parenteral administration ofthe compound of Formula (I) is preferred. An intravenous infusion of thecompound in 5% dextrose in water or normal saline, or a similarformulation with suitable excipients, is most effective, although anintramuscular bolus injection is also useful. Typically, the parenteraldose will be about 0.01 to about 100 mg/kg; preferably between 0.1 and20 mg/kg, in a manner to maintain the concentration of drug in theplasma at a concentration effective to inhibit cathepsin K. The compoundis administered one to four times daily at a level to achieve a totaldaily dose of about 0.4 to about 400 mg/kg/day. The precise amount ofthe inventive compound which is therapeutically effective, and the routeby which such compound is best administered, is readily determined byone of ordinary skill in the art by comparing the blood level of theagent to the concentration required to have a therapeutic effect.

Prodrugs of the compound of the present invention may be prepared by anysuitable method. Where the prodrug moiety is a ketone functionality,specifically ketals and/or hemiacetals, the conversion may be effectedin accordance with conventional methods.

The compound of this invention may also be administered orally to thepatient, in a manner such that the concentration of drug is sufficientto inhibit bone resorption or to achieve any other therapeuticindication as disclosed herein. Typically, a pharmaceutical compositioncontaining the compound is administered at an oral dose of between about0.1 to about 50 mg/kg in a manner consistent with the condition of thepatient. Preferably the oral dose would be about 0.5 to about 20 mg/kg.

No unacceptable toxicological effects are expected when compounds of thepresent invention are administered in accordance with the presentinvention.

Biological Assays

The compound of this invention may be tested in one of severalbiological assays to determine the concentration of the compound whichis required to have a given pharmacological effect.

Determination of Cathepsin K Proteolytic Catalytic Activity

All assays for cathepsin K were carried out with human recombinantenzyme. Standard assay conditions for the determination of kineticconstants used a fluorogenic peptide substrate, typicallyCbz-Phe-Arg-AMC, and were determined in 100 mM Na acetate at pH 5.5containing 20 mM cysteine and 5 mM EDTA. Stock substrate solutions wereprepared at concentrations of 10 or 20 mM in DMSO with 20 μM finalsubstrate concentration in the assays. All assays contained 10% DMSO.Independent experiments found that this level of DMSO had no effect onenzyme activity or kinetic constants. All assays were conducted atambient temperature. Product fluorescence (excitation at 360 nM;emission at 460 nM) was monitored with a Perceptive Biosystems CytofluorII fluorescent plate reader. Product progress curves were generated over20 to 30 minutes following formation of AMC product.

Inhibition Studies

Potential inhibitors were evaluated using the progress curve method.Assays were carried out in the presence of variable concentrations oftest compound. Reactions were initiated by addition of enzyme tobuffered solutions of inhibitor and substrate. Data analysis wasconducted according to one of two procedures depending on the appearanceof the progress curves in the presence of inhibitors. For thosecompounds whose progress curves were linear, apparent inhibitionconstants (K_(i,app)) were calculated according to equation 1 (Brandt etal., Biochemistry, 1989, 28, 140):

v=V _(m) A/[K _(a)(l+I/K _(i, app))+A]  (1)

where v is the velocity of the reaction with maximal velocity V_(m), Ais the concentration of substrate with Michaelis constant of K_(a), andl is the concentration of inhibitor.

For those compounds whose progress curves showed downward curvaturecharacteristic of time-dependent inhibition, the data from individualsets was analyzed to give k_(obs) according to equation 2:

[AMC]=v _(ss) t+(v ₀ −v _(ss))[l−exp (−k _(obs) t)]/k_(obs)  (2)

where [AMC] is the concentration of product formed over time t, v₀ isthe initial reaction velocity, and v_(ss) is the final steady staterate. Values for k_(obs) were then analyzed as a linear function ofinhibitor concentration to generate an apparent second order rateconstant (k_(obs)/inhibitor concentration or k_(obs)/[I]) describing thetime-dependent inhibition. A complete discussion of this kinetictreatment has been fully described (Morrison et al., Adv. Enzymol.Relat. Areas Mol. Biol., 1988, 61, 201).

One skilled in the art would consider any compound with a K_(i) of lessthan 50 micromolar to be a potential lead compound. Preferably, thecompounds used in the method of the present invention have a K_(i) valueof less than 1 micromolar. Most preferably, said compounds have a K_(i)value of less than 100 nanomolar.

Human Osteoclast Resorption Assay

Aliquots of osteoclastoma-derived cell suspensions were removed fromliquid nitrogen storage, warmed rapidly at 37° C. and washed ×1 inRPMI-1640 medium by centrifugation (1000 rpm, 5 min at 4° C.). Themedium was aspirated and replaced with murine anti-HLA-DR antibody,diluted 1:3 in RPMI-1640 medium, and incubated for 30 minutes on ice.The cell suspension was mixed frequently.

The cells were washed ×2 with cold RPMI-1640 by centrifugation (1000rpm, 5 min at 4° C.) and then transferred to a sterile 15 mL centrifugetube. The number of mononuclear cells were enumerated in an improvedNeubauer counting chamber.

Sufficient magnetic beads (5/mononuclear cell), coated with goatanti-mouse IgG, were removed from their stock bottle and placed into 5mL of fresh medium (this washes away the toxic azide preservative). Themedium was removed by immobilizing the beads on a magnet and is replacedwith fresh medium.

The beads were mixed with the cells and the suspension was incubated for30 minutes on ice. The suspension was mixed frequently. The bead-coatedcells were immobilized on a magnet and the remaining cells(osteoclast-rich fraction) were decanted into a sterile 50 mL centrifugetube. Fresh medium was added to the bead-coated cells to dislodge anytrapped osteoclasts. This wash process was repeated ×10. The bead-coatedcells were discarded.

The osteoclasts were enumerated in a counting chamber, using alarge-bore disposable plastic pasteur pipette to charge the chamber withthe sample. The cells were pelleted by centrifugation and the density ofosteoclasts adjusted to 1.5×10⁴/mL in EMEM medium, supplemented with 10%fetal calf serum and 1.7g/litre of sodium bicarbonate. 3 mL aliquots ofthe cell suspension (per treatment) were decanted into 15 mL centrifugetubes. These cells were pelleted by centrifugation. To each tube 3 mL ofthe appropriate treatment was added (diluted to 50 μM in the EMEMmedium). Also included were appropriate vehicle controls, a positivecontrol (87MEM1 diluted to 100 ug/mL) and an isotype control (IgG2adiluted to 100 ug/mL). The tubes were incubated at 37° C. for 30minutes.

0.5 mL aliquots of the cells were seeded onto sterile dentine slices ina 48-well plate and incubated at 37° C. for 2 hours. Each treatment wasscreened in quadruplicate. The slices were washed in six changes of warmPBS (10 mL/well in a 6-well plate) and then placed into fresh treatmentor control and incubated at 37° C. for 48 hours. The slices were thenwashed in phosphate buffered saline and fixed in 2% glutaraldehyde (in0.2 M sodium cacodylate) for 5 minutes, following which they were washedin water and incubated in buffer for 5 minutes at 37° C. The slices werethen washed in cold water and incubated in cold acetate buffer/fast redgarnet for 5 minutes at 4° C. Excess buffer was aspirated, and theslices were air dried following a wash in water.

The TRAP positive osteoclasts were enumerated by bright-field microscopyand were then removed from the surface of the dentine by sonication. Pitvolumes were determined using the Nikon/Lasertec ILM21W confocalmicroscope.

EXAMPLES

In the following synthetic examples, unless otherwise indicated, all ofthe starting materials were obtained from commercial sources. Withoutfurther elaboration, it is believed that one skilled in the art can,using the preceding description, utilize the present invention to itsfullest extent. These Examples are given to illustrate the invention,not to limit its scope. Reference is made to the claims for what isreserved to the inventors hereunder.

Example 1 Preparation of Quinoxaline-2-Carboxylic Acid{(S)-3-methyl-1-[(2,2′,4-trideuterio)-3-oxo-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide

a.) Allyl-pent-4-enyl-carbamic Acid Benzyl Ester

To a suspension of NaH (1.83 g, 76.33 mmol of 90% NaH) in DMF was addedallyl-carbamic acid benzyl ester (7.3 g, 38.2 mmol) in a dropwisefashion. The mixture was stirred at room temperature for approximately10 minutes whereupon 5-bromo-1-pentene (6.78 mL, 57.24 mmol) was addedin a dropwise fashion. The reaction was heated to 40° C. forapproximately 4 hours whereupon the reaction was partitioned betweendichloromethane and water. The organic layer was washed with water(2×'s), brine, dried (MgSO₄), filtered and concentrated. Columnchromatography of the residue (10% ethyl acetate:hexanes) provided 10.3grams of the title compound as an oil: MS(EI) 260 (M+H⁺).

b.) 2,3,4,7-Tetrahydro-azepine-1-carboxylic Acid Benzyl Ester

To a solution of compound of Example 1a (50 g) in dichloromethane wasadded bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride(5.0 g). The reaction was heated to reflux until complete as determinedby TLC analysis. The reaction was concentrated in vacuo. Columnchromatography of the residue (50% dicbloromethane:hexanes) gave 35 g ofthe title compound: MS(EI) 232 (M+H⁺).

c.) 8-Oxa-3-aza-bicyclo[5.1.0]octane-3-carboxylic Acid Benzyl Ester

To a solution of the compound of Example 1b (35 g, 1.5 mol) in CH₂Cl₂was added m-CPBA (78 g, 0.45 mol). The mixture was stirred overnight atroom temperature whereupon it was filtered to remove the solids. Thefiltrate was washed with water and saturated NaHCO₃ (several times). Theorganic layer was dried (MgSO₄), filtered and concentrated to give 35 gof the title compound which was of sufficient purity to carry on to thenext step: MS(EI) 248 (M+H⁺), 270 (M+Na⁺).

d. 4-Azido-3-hydroxy-azepane-1-carboxylic Acid Benzyl Ester

To a solution of the epoxide from Example 1c (2.0 g, 8.1 mmol) inmethanol:water (8:1 solution) was added NH₄Cl (1.29 g, 24.3 mmol) andsodium azide (1.58 g, 24.30 mmol). The reaction was heated to 65-75° C.until complete consumption of the starting epoxide was observed by TLCanalysis. The majority of the solvent was removed in vacuo and theremaining solution was partitioned between ethyl acetate and pH 4buffer. The organic layer was washed with sat. NaHCO₃, water, brinedried (MgSO₄), filtered and concentrated. Column chromatography (20%ethyl acetate:hexanes) of the residue provided 1.3 g of the titlecompound: MS(EI) 291 (M+H⁺) plus 0.14 g oftrans-4-hydroxy-3-azido-hexahydro-1H-azepine

e.) 4-Amino-3-hydroxy-azepane-1-carboxylic Acid Benzyl Ester

To a solution of the azido alcohol of Example 1d (1.1 g, 3.79 mmol) inmethanol was added triethyamine (1.5 mL, 11.37 mmol) and1,3-propanedithiol (1.1 mL, 11.37 mmoL). The reaction was stirred untilcomplete consumption of the starting material was observed by TLCanalysis whereupon the reaction was concentrated in vacuo. Columnchromatography of the residue (20% methanol:dichloromethane) provided0.72 g of the title compound: MS(EI) 265 (M+H⁺).

f.)4-((S)-2-tert-Butoxycarbonylamino-4-methyl-pentanoylamino)-3-hydroxy-azepan-1-carboxylicAcid Benzyl Ester

To a solution of the amino alcohol of Example 1e (720 mg, 2.72 mmol) inCH₂Cl₂ was added EDC (521 mg), HOBt (368 mg) and N-Boc-leucine (630 mg).The reaction was maintained at room temperature until completeconsumption of the starting material was observed by TLC analysis. Thereaction was diluted with ethyl acetate and washed with 1N HCl, sat.K₂CO₃, water, brine, dried (MgSO₄), filtered and concentrated. Columnchromatography of the residue (3% methanol:dichloromethane) gave 1.0 gof the title compound: MS(EI) 478 (M+H⁺).

g.) [(S)-1-(3-Hydroxy-azepan-4-ylcarbamoyl)-3-methyl-butyl]-carbamicAcid tert Butyl Ester

To a solution of the compound of Example 1f (1.0 g) and 10% Pd/C(catalytic) in ethyl acetate:methanol (2:1 solution) was attached aballoon of hydrogen. The reaction was stirred until complete consumptionof the starting material was observed by TLC analysis. The reaction wasfiltered to remove the catalyst and the filtrate was concentrated toprovide 0.82 g of the title compound: MS(EI) 344 M+H⁺).

h.){(S)-1-[3-Hydroxy-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-3-methyl-butyl}-carbamicAcid tert-butyl Ester

Generation of 2-pyridinesulfonyl chloride-N-oxide: To a 0° C. solutionof 2-mercaptopyridine-N-oxide (2.23 g, 17.55 mmol) in 9M HCl (33 mL) wasbubbled chlorine gas for approximately 90 minutes. The dissolvedchlorine was removed under vacuum at 0° C.

To a solution of[(S)-1-(3-hydroxy-azepan-4-ylcarbamoyl)-3-methyl-butyl]-carbamic acidtert butyl ester of Example 1g (2.5 g, 7.28 mmol) in CH₂Cl₂ (100 mL) andsat. NaHCO₃ (400 mL) was added the solution of 2-pyridinesulfonylchloride-N-oxide (27 mL, 102 mg/mL) dropwise in portions. As theaddition proceeds additional sat. NaHCO₃ is added in order to maintainthe pH at approximately 8-9. Upon complete addition of thesulfonylchloride the reaction is stirred for an additional hourwhereupon the organic layer was removed and washed with brine. Theorganic layer was evaporated and the residue chromatographed (5%methanol:dichloromethane) to provide 2.5 g of the title compound: MS(EI) 500 (M+H⁺).

i.) (S)-2-Amino-4-methyl-pentanoicAcid-[3-hydroxy-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-yl]-amide

To a solution of{(S)-1-[3-hydroxy-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-3-methyl-butyl}-carbamicacid tert-butyl ester of Example 1h (2.0 g) in methanol (20 mL) wasadded 4 M HCl in dioxane (20 mL). The reaction was stirred at roomtemperature for 1.5 hours whereupon it was concentrated to provide 1.8 gof the title compound: MS (EI) 400 (M+H⁺).

j.) Quinoxaline-2-carboxylic Acid{(S)-3-methyl-1-[3-hydroxy-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide

To a solution of the compound of Example 1i (320 mg, 0.73 mmol) inCH₂Cl₂ was added triethylamine (0.15 mL, 1.09 mmol), EDC (140 mg, 0.73mmol), HOBt (99 mg, 0.73 mmol) and quinoxaline-2-carboxylic acid (127mg, 0.73 mmol). The reaction was stirred until complete by TLC analysis.Workup and column chromatography of the residue gave the title compound:MS(EI) 556 (M⁺).

k.) Quinoxaline-2-carboxylic Acid{(S)-3-methyl-1-[3-oxo-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide

To a solution of the alcohol of Example 1j (0.28 g, 0.5 mmol) in DMSOwas added TEA (0.42 mL, 3.0 mmol) and pyridine sulfur trioxide complex(238 mg, 1.5 mmol). The reaction was stirred at room temperature forapproximately 2 hours whereupon it was partitioned between ethyl acetateand water. The organic layer was washed with brine, dried, filtered andconcentrated. Column chromatography of the residue (10% CH₃OH:CH₂Cl₂)provided 231 mg of the title compound as a mixture of diastereomers: ¹HNMR (CDCl₃): ¹H NMR (CDCl₃): δ 1.0 (m, 6H), 1.5-2.1 (m, 5H), 2.2 (m,2H), 2.7 (m, 1H), 3.8 (q, 1H). 4.0 (m, 1H), 4,5 (t, 1H), 4.7 (m, 1H),5.0 (m, 1H), 7.4-7.5 (m, 2H), 7.9 (m, 1H), 8.0-8.4 (m, 4H), 9.6 (d, 1H);MS(EI): 554 (M⁺, 100%).

l.) Quinoxaline-2-carboxylic Acid{(S)-3-methyl-1-[(2,2′,4-trideuterio)-3-oxo-1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide

To a solution of quinoxaline-2-carboxylic acid{(S)-3-methyl-1-[3-oxo-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide(0.03 g) in D₂O:CD₃OD (0.4:4 mL) was added triethylamine (0.04 mL). Thereaction was heated to reflux for 2 hours whereupon it was concentratedand dried under vacuum. The residue was then redissolved in the samemixture and heated to reflux overnight. The reaction was concentratedand the residue purified by column chromatography to provide the titlecompound. The diastereomers are separated by HPLC.

The above specification and Example fully disclose how to make and usethe compound of the present invention. However, the present invention isnot limited to the particular embodiments described hereinabove, butincludes all modifications thereof within the scope of the followingclaims. The various references to journals, patents and otherpublications which are cited herein comprise the state of the art andare incorporated herein by reference as though fully set forth.

What is claimed is:
 1. A compound according to Formula (I):

(quinoxaline-2-carboxylic acid{(S)-3-methyl-1-[(2,2′,4-trideuterio)-3-oxo-1-(1-oxy-pyridine-2-sulfonyl)-azepan-4-ylcarbamoyl]-butyl}amide),or a pharmaceutically acceptable salt, hydrate or solvate thereof.
 2. Apharmaceutical composition comprising a compound according to claim 1and a pharmaceutically acceptable carrier, excipient or diluent.
 3. Amethod of inhibiting a protease selected from the group consisting of acysteine protease and a serine protease, comprising administering to apatient in need thereof an effective amount of a compound according toclaim
 1. 4. A method according to claim 3 wherein said protease is acysteine protease.
 5. A method according to claim 4 wherein saidcysteine protease is cathepsin K.
 6. A method of treating a diseasecharacterized by bone loss comprising inhibiting said bone loss byadministering to a patient in need thereof an effective amount of acompound according to claim
 1. 7. A method according to claim 6 whereinsaid disease is osteoporosis.
 8. A method according to claim 6 whereinsaid disease is periodontitis.
 9. A method according to claim 6 whereinsaid disease is gingivitis.
 10. A method of treating a diseasecharacterized by excessive cartilage or matrix degradation comprisinginhibiting said excessive cartilage or matrix degradation byadministering to a patient in need thereof an effective amount of acompound according to claim
 1. 11. A method according to claim 10wherein said disease is osteoarthritis.
 12. A method according to claim10 wherein said disease is rheumatoid arthritis.
 13. A process for thesynthesis of the compound of Formula (I):

comprising the steps: (a) of oxidizing the compound of Formula (II):

with an oxidant to provide the protonated isomer of the compound ofFormula (I) as a mixture of diastereomers; (b) deuterating theprotonated isomer to provide the compound of Formula (I) as a mixture ofdiastereomers.
 14. The process of claim 13 wherein the oxidant is sulfurtrioxide pyridine complex in DMSO and triethylamine.
 15. The process ofclaim 13 wherein the deuterating agent is CD₃OD:D₂O (10:1) intriethylamine.
 16. The process of claim 13 further comprising the stepof separating the diasteromers by a separating means.
 17. The process ofclaim 16 wherein said separating means is high presssure liquidchromatography (HPLC).